The toxicity of antifolates for cells and the consequences of antifolate inhibition of DNA synthesis has been a central focus of our work over the past two years. Our studies have examined the conditions required for establishing and maintaining the metabolic toxicity of MTX in WIL-2 cells and leukemic cells. As a result of these studies we have established a rationale for utilizing low doses of MTX or for complementary use of low concentrations of the lipid soluble antifolate, DDMP for maintenance of MTX toxicity. Such protocols may increase the selectivity of MTX based regimens, and have special efficacy for treatment of tumors of the central nervous system. Our studies have also extended the utility of assays for antifolate activity based on UdR incorporation into DNA.